The O, S, and Sr isotopic compositions were determined for 17 samples of gypsum from the sheeted dike complex of the Macquarie Island ophiolite in order to trace chemical reactions during hydrothermal recharge. Macquaire Island is a sliver of N-MORB ocean crust tectonically exposed on the Australian-Pacific plate boundary. The gypsums have d¹⁸O values of 12.5-14.4‰,which preclude meteoric water and confirm their formation, probably as anhydrite, from seawater fluids beneath the seafloor. The gypsums have elevated d³⁴S values (26.2-29.0‰) compared to seawater (21‰), as the result of sulfate reduction. The minerals formed at temperatures of 137-158°C if formed in oxygen isotopic equilibrium with normal seawater. At these temperatures, inorganic sulfate reduction is kinetically inhibited, and fractionation between sulfate and sulfide is too large (~35‰) to yield ³⁴ S-enriched hydrothermal sulfide in the dike wallrocks. The gypsums have ⁸⁷Sr/⁸⁶ Sr ratios of 0.70446-0.70524 reflecting highly reacted solutions compared to modern seawater (0.7091). Kinetic effects on oxygen isotopic exchange in sulfate or ¹⁸O-depletions of the fluid resulting from water-rock interations would decrease the calculated formation temperatures of gypsum, and could put temperatures in the range where microbial sulfate reduction could occur. In this scenario, formation waters residing in the volcanic section at temperatures of ~100°C underwent significant seawater-rock interaction and sulfate in solution was reduced to sulfide by microbial activity. Supporting evidence includes negative d³⁴S values of sulfide minerals in the lavas, consistent with microbial sulfate reduction there. Tectonic activity at the spreading ridge allowed ingress of evolved formation waters from the lavas into hot dikes, and resulted in precipitation of anhydrite during heating of these fluids. Anhydrite was hydrated to gypsum as the dikes cooled during subsequent aging of the crust. Alternatively, anhydrite may have formed within the dikes by a complex process of mixing high-temperature hydrothermal fluids with cooler seawater fluids.